Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

• MeSH
• Cell Adhesion MeSH
• Flagella * metabolism   MeSH
• Insect Vectors parasitology   MeSH
• Insect Proteins metabolism   genetics   MeSH
• Host-Parasite Interactions MeSH
• Leishmania * physiology   genetics   metabolism   MeSH
• Leishmaniasis parasitology   transmission   MeSH
• Protozoan Proteins metabolism   genetics   MeSH
• Psychodidae * parasitology   MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insect Proteins MeSH
• Protozoan Proteins MeSH

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.

• Keywords
• Leishmania, allelic selection, sand fly infection,
• MeSH
• Leishmania donovani * MeSH
• Humans MeSH
• Mutation MeSH
• DNA Repair genetics   MeSH
• Oxidative Stress genetics   MeSH
• Psychodidae * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.

• Keywords
• Leishmania, flagellar pocket, morphogenesis, pathogenicity,
• MeSH
• Cell Membrane metabolism   MeSH
• Cilia genetics   physiology   ultrastructure   MeSH
• Gene Deletion MeSH
• Endocytosis MeSH
• Flagella genetics   physiology   ultrastructure   MeSH
• Host-Parasite Interactions MeSH
• Leishmania genetics   pathogenicity   physiology   ultrastructure   MeSH
• Intercellular Junctions MeSH
• Mice MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Psychodidae parasitology   MeSH
• Virulence genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protozoan Proteins MeSH

BACKGROUND: The peritrophic matrix (PM) is an acellular chitin-containing envelope which in most blood sucking insects encloses the ingested blood meal and protects the midgut epithelium. Type I PM present in sand flies and other blood sucking batch feeders is secreted around the meal by the entire midgut in response to feeding. Here we tested the hypothesis that in Sergentomyia schwetzi the PM creates a physical barrier that prevents escape of Leishmania parasites from the endoperitrophic space. METHODOLOGY/PRINCIPAL FINDINGS: Morphology and ultrastructure of the PM as well the production of endogenous chitinase in S. schwetzi were compared with three sand fly species, which are natural vectors of Leishmania. Long persistence of the PM in S. schwetzi was not accompanied by different morphology or decreased production of chitinase. To confirm the role of the PM in refractoriness of S. schwetzi to Leishmania parasites, culture supernatant from the fungus Beauveria bassiana containing chitinase was added to the infective bloodmeal to disintegrate the PM artificially. In females treated with B. bassiana culture supernatants the PM was weakened and permeable, lacking multilayered inner structure; Leishmania colonized the midgut and the stomodeal valve and produced metacyclic forms. In control females Leishmania infections were lost during defecation. CONCLUSIONS/SIGNIFICANCE: Persistence of the PM till defecation of the bloodmeal represents an important factor responsible for refractoriness of S. schwetzi to Leishmania development. Leishmania major as well as L. donovani promastigotes survived defecation and developed late-stage infections only in females with PM disintegrated artificially by B. bassiana culture supernatants containing exogenous chitinase.

• MeSH
• Insect Vectors parasitology   physiology   ultrastructure   MeSH
• Rabbits MeSH
• Leishmania major physiology   MeSH
• Psychodidae parasitology   physiology   ultrastructure   MeSH
• Digestive System parasitology   ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. RESULTS: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. CONCLUSIONS: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

• Keywords
• Leishmania amazonensis, Lipophosphoglycan, Lutzomyia longipalpis, Lutzomyia migonei, Phlebotomus papatasi, Vector-parasite interaction,
• MeSH
• Glycosphingolipids analysis   MeSH
• Leishmania chemistry   growth & development   isolation & purification   MeSH
• Humans MeSH
• Psychodidae parasitology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycosphingolipids MeSH
• lipophosphonoglycan MeSH Browser